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Krannert Institute of Cardiology and Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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A method is described
for freezing thin strips of smooth muscle by replacing physiological
saline in the muscle chamber with cold organic solvent in <100 ms.
Calculations suggest that, with a perfectly stirred boundary at the
tissue surface, freezing could occur within ~15 ms at the center of a
200-µm-thick piece of tissue by use of acetone coolant at 
78.5°C
and in approximately half the time with either isopentane at its
freezing point (160°C) or aluminum chilled with liquid nitrogen.
Myosin light chain phosphorylation in muscles frozen with cold acetone
began to rise ~200 ms earlier than force and increased at a much more
rapid rate. The difference in onsets of the two processes reflects the
delay in arresting phosphorylation plus two lags associated with force
generation, attachment of phosphorylated bridges followed by force
generating movements of the attached bridges. The much more rapid rise
of phosphorylation, once it began, suggests that most of this delay is
due to physiological lags and not to slow arrest of metabolism.
tissue freezing; myosin light chain phosphorylation; myosin light chain kinase; arrest of metabolism
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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MANY INVESTIGATIONS OF PHYSIOLOGICAL process in muscle and other tissues require accurately timed, rapid freezing to stop enzymatic reactions for assay of reactants. We describe here a simple and inexpensive apparatus capable of rapidly exchanging very cold organic solvent for warm saline in a muscle bath with precise timing of the exchange. Although we use this technique for freezing smooth muscle tissue in preparation for assays of myosin light chain phosphorylation, it seems likely that the apparatus could be used for rapidly freezing many types of solid tissue.
A lag between the onsets of phosphorylation and force is used to estimate the upper limit of the time over which metabolic processes would be arrested in the belief that phosphorylation is required for myosin activation and force generation. This lag reflects two processes in series, one artifactual and the other physiological. Because phosphorylation continues for a time after freezing is initiated, the level of phosphorylation at the time of freezing will appear higher in relation to force measured at the same instant. This delay in the arrest of metabolism creates the appearance of a delay between phosphorylation and force. There must also be genuine delays between the time a myosin molecule is phosphorylated and the time it attaches to actin and generates force. The results below suggest that most of the lag between phosphorylation and force is due to physiological processes and not to the delay in the arrest of metabolism.
Finally, we describe calculations of the time required to freeze tissue and of the temperature profiles across tissues, both for our own apparatus and for that used by others. We do this both to estimate the cooling time in the tissue and to compare our technique with other methods.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Principle of the Method
Muscles were studied in a narrow vertical chamber open at the top so that force could be recorded with a transducer above the chamber. Events were controlled by a computer also used to record force, so that the time of freezing was precisely coordinated with the initiation of stimulation and force recording. Solution changes were accomplished with three separate "pumps" made from disposable syringes (Fig. 1A), driven by vacuum, and activated by computer-controlled solenoid valves in the vacuum lines driving the pumps. The first of these pumps was coupled in a way that the application of vacuum (S in Fig. 1A) withdrew the warm saline and a limited quantity of air from the chamber. The other two were coupled in the reverse manner, so that application of vacuum injected acetone at78°C into the chamber. The first made a
rapid injection to just fill the chamber and the second caused a slow injection of more cold acetone over several seconds. As this continued, slower flow drew additional heat from the muscle and chamber and allowed the acetone to be drawn away by suction from the top of the
chamber without spilling.
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Muscle Chamber
A diagram of the apparatus is shown in Fig. 1B. The vertical chamber was milled from a 50 × 50 × 13-mm-thick block of delrin (obtained from Small Parts, Miami Lakes, FL) to form the bottom and two sides. The back and front of the chamber were of 1-mm-thick glass, cut from microscope slides, clamped to the delrin. The glass allowed the muscle to be viewed in the chamber and permitted the simple method of measuring the speed of solution changes using the optical methods described below. The delrin block was milled to yield a vertical chamber, 28 mm high × 7.5 mm wide. Insets 4.5 mm deep were milled around the slot both back and front, and the glass pieces were clamped in these insets, leaving a 4-mm front-to-back dimension for the muscle chamber. This reduction of the thicker block to a narrow chamber reduced the chamber volume while retaining sufficient thickness around the edge to allow tubing connections to be placed in the perimeter of the delrin block.Three ports converged in the bottom of the chamber. Warm saline was carried to the bottom of the chamber from a port entering at one side (R in Fig. 1B). Cold acetone was injected into the bottom of the chamber through a port on the opposite side. A wide port projecting vertically downward was used to withdraw the saline immediately before freezing (W in Fig. 1B). A bent length of 20-gauge stainless steel tubing (not shown) projected into the top of the chamber to carry off both saline and acetone that rose to the top during periods of continuous flow. Blackened platinum electrodes were fastened to the delrin sides of the chamber. The tubing connected to the ports was sufficiently flexible that the chamber could be lowered for mounting and removing the muscle. The entire block assembly was mounted on a micromanipulator (M2 in Fig. 1B) to maintain accurate positioning as the chamber was moved up and down.
Muscle Mounts
Strips of tracheal smooth muscle measuring ~0.1-0.2 mm × 1.5 mm × 7 mm long were held at both ends by aluminum foil "T-clips" (4). Holes in the clips were passed over hooks to attach the muscle to the apparatus. The upper hook, fashioned from a 7-cm length of 0.125-mm-diameter stainless steel wire, was used to connect the muscle to a photoelectric force transducer (2) located above the chamber (FT in Fig. 1B). The clip at the bottom end of the muscle was hooked to a J-shaped piece of hypodermic tubing firmly connected to the transducer by a rack and pinion used to adjust muscle length. Thus the muscle attachments were mechanically separated from the chamber to allow the block containing the chamber to be lowered so that the muscle could be mounted and removed quickly. Muscle length was adjusted by altering the height of the transducer with a micromanipulator to which it was attached (M1 in Fig. 1B).Syringe Pumps
Fixed capacity syringe pumps (Fig. 1A) were used to ensure that exact quantities of fluid were withdrawn and injected into the trough. These were made from coupled pairs of plastic syringes. Using a wide-bore 50-ml syringe to apply vacuum and a narrow-bore 10-ml syringe to move solution provided an ~3.5-fold pneumatic advantage. For the withdrawal pump, the barrels and plungers of the two syringes were connected. Withdrawing air from the large syringe caused the plunger of the small syringe to withdraw fluid and air from the chamber. For injection pumps, the barrel of each syringe was connected to the plunger of the opposing syringe. Withdrawal of air from the large syringe then caused the plunger of the small syringe to expel air. Two pumps were used for injection. A rapid pump was used to fill the chamber with coolant in ~100 ms, and a second pump was used to inject additional coolant at a slower rate without spillage. To slow the rate of the second pump, a 25-gauge 1.5-in.-long hypodermic needle was inserted in the vacuum line. This reduced the travel time of the syringe barrels from ~100 ms to ~4 s.Flow Control
Vacuum was obtained from a laboratory wall outlet. A vacuum reservoir was provided by a suction flask that also served as a waste trap for fluid drawn off from the top of the chamber. Several seconds before freezing, saline flow to the trough was halted. The pumps were activated by the computer ~200 ms before freezing was to occur.Before injection, the acetone was contained in a sealed plastic Falcon
tube immersed in an insulated cup (CC in Fig. 1B) containing a mixture of dry ice and acetone, so that the entire contents of the
cup were close to the sublimation point of dry ice (78°C). Air
injected into the top of the Falcon tube forced cold acetone through a
tube extending from the bottom of the tube to the chamber through a
three-way stopcock. Until a few seconds before the injection, the
stopcock was turned so that the Falcon tube was vented to the
atmosphere and the bottom of the chamber was sealed. It was then turned
so that the tubing from the Falcon tube led into the chamber.
Vacuum flow was controlled with solenoid valves obtained from General Valve (East Hanover, NJ). Experiment control and recording were accomplished with an IBM-compatible computer equipped with a Tecmar (Solon, OH) Labmaster interface board and the SALT software package (3).
Measuring Solution Changes
A beam of light ~5 mm wide derived from a laser diode was shone through the chamber at a 5-mm-diameter photodiode on the opposite side. The face of the diode was masked by an oblong slit, 1 mm wide × 3 mm high, to provide a signal that varied in a quasi-linear manner as a meniscus of dye flowed up or down the chamber. The rate of fluid flow was assessed from this signal when dye was moved in and out of the chamber.Light Chain Phosphorylation
Myosin light chain phosphorylation was measured by quantitative densitometry of films exposed to chemiluminescent Western blots of light chains (8).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Timing of Solution Changes
Figure 2 is a recording of the output of a photodiode illuminated by a light beam passing through a 3-mm-high slit at the approximate vertical center of the muscle chamber. Digital data points were recorded every millisecond. As shown, there was an ~100-ms delay between the time the withdrawal pump was activated (A) and the time that diode output began to rise. The delay arises mainly from the time required for sufficient air evacuation from the syringe pump to overcome the sticking friction between plunger and barrel and partly from the time needed to reduce air pressure in the line connecting the pump to the chamber.
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The fall of the meniscus through the 3-mm-high light beam was complete in ~6 ms. This time suggests that fluid was removed from the 28-mm-high chamber in ~55 ms.
Figure 2 also shows the timing of the subsequent fall in the diode output when dye was injected back into the chamber. The meniscus began to pass through the light beam ~190 ms after activation of the injection pumps (B in Fig. 2) and completed its passage through the light beam in ~7 ms. Both the delay in the appearance of movement and the rate of meniscus flow are somewhat slower than with withdrawal. These slower times are expected from the greater volume of air behind the injected fluid. The 7-ms time taken for the meniscus to pass through the 3-mm-light beam suggests that the fluid would completely inundate a 7.5-mm length of muscle in ~18 ms.
Completeness of Solution Changes
A bolus of air precedes the flow of coolant into the bottom of the chamber. If this air flow begins before removal of warm saline is complete, saline removal is interrupted. Such incomplete emptying of the chamber was recognized by pieces of ice mixed with the cold acetone. It was found that such complications were avoided when the injection pump was activated ~10 ms after the withdrawal pump so that cold acetone reached the muscle ~100 ms after the saline had been drawn away. This timing for freezing was used for all tissues assayed for myosin light chain phosphorylation.Freezing Rate
The delay between the application of cold acetone and sufficient cooling was estimated in two ways. One was a calculation of the times required to cool the center of the muscle from 37 to 0°C and then to freeze the tissue. The other was a measure of the interval between the first appearance of myosin light chain phosphorylation measured in frozen muscles and the onset of force development.Numerical estimates of freezing times.
A numerical method for calculating cooling and freezing rates in muscle
tissue is described in the APPENDIX, and the results are
summarized in Fig. 3. The values plotted
are the times taken to cool a 200-µm-thick muscle from 37 to 0°C
and then freeze it. Calculations are made for three separate
conditions: acetone at 78°C; isopentane at its freezing point,
160°C; and aluminum hammers at the boiling point of liquid
nitrogen, 196°C. These calculations indicate that, under ideal
conditions of perfectly stirred boundaries, freezing of the tissue
center in acetone at 78°C would occur in ~15 ms. The calculations
further show that freezing should be about twice as fast with either
isopentane at 160°C or aluminum hammers at 196°C. These results
are used below in discussing the relative merits of the different
techniques.
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Lag between phosphorylation and force.
Because myosin phosphorylation is required for smooth muscle activation
(1, 6, 9), the lag measured between the first appearance
of phosphorylation and the onset of force development includes both the
time required to freeze the muscle plus the delay between activation of
myosin and the appearance of force. Thus the lag between the time of
freezing when phosphorylation had just risen above the baseline value
and the first appearance of force gives an upper limit for the time
taken to cool the muscle sufficiently to stop the phosphorylation. To
measure this interval, muscles were stimulated to produce 12-s tetani
every 5 min, and force was recorded digitally at 100-ms intervals
throughout the tetanus. As shown in Fig.
4, at 37°C, there was a latency period of ~400-500 ms between the onset of stimulation and the first appearance of force. In the resting state, immediately before stimulation, there was a slight degree of phosphorylation (3.8%). This
low level was approximately doubled in muscles frozen at 300 ms after
the onset of stimulation and was increased by approximately sevenfold
(to 28%) 500 ms later, 800 ms after the onset of stimulation. This
last value was a little more than half the maximum value measured in
muscles frozen at later times. By contrast, force had risen to only
~2% of its full value at the same time and to only ~12% of its
full value by 1.3 s (Fig. 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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A technique is described for rapidly freezing tissue in a precisely timed manner. Comparing the first appearance of phosphorylation with the first appearance of force shows that the two are separated by ~200 ms. Because phosphorylation is likely to continue for at least a brief period after the coolant reaches the muscle, some of this lag is due to the time taken to freeze the tissue. The remainder is due to a physiological lag between the chemical change in the myosin and the appearance of force. Two processes that are likely to contribute to this physiological lag are the attachment of myosin cross bridges to thin filaments and force-generating movements of the attached cross bridges. This consideration raises the question of the relative contributions of the two processes to the overall delay measured. The very rapid rise of phosphorylation compared with force suggests that much of the lag is due to the physiological delay between phosphorylation and force generation. If this were not the case, if force developed very soon after myosin was phosphorylated, the rise of force would parallel the rise of phosphorylation and simply be shifted in time by an amount equivalent to the freezing lag.
Relationship to Earlier Work
The present apparatus combines three important characteristics: speed, accurate timing, and simplicity. Other methods of freezing usually optimize only one or two of these properties at the expense of the others. For example, the most prevalent method of freezing muscle is to lower the chamber containing physiological saline and to replace it with a container of freezing solution, often by hand. The greatest virtue of this method is simplicity. There is likely to be a substantially greater lag between the removal of the warm saline and the application of cold solution, but, once begun, the freezing is likely to be nearly as fast as the present method, provided that the freezing solution does not vaporize, as does liquid nitrogen. When a substance, such as liquid nitrogen, that boils at temperatures much below 37°C is used for freezing, there is likely to be an insulating layer of gas surrounding the tissue. Because of the relative slowness of the solution change and inaccuracy in the timing of events, this method is best used for measuring steady-state reactant concentrations or concentrations of reactants that change slowly, for which precise timing is not required.At the other extreme, Gilbert et al. (5) developed a method for squashing muscles between large aluminum hammers cooled by liquid nitrogen. As with the present system, this method is both rapid and precisely timed. The relative merits of the two systems will therefore be compared.
Comparison of freezing techniques. The two advantages of the present technique are 1) it is inexpensive and simple, being made from readily available laboratory apparatus; and 2) it can be used safely in conjunction with delicate instruments, such as force transducers, that are rigidly attached to the tissue. The three advantages of the cold-hammer freezing method are 1) the hammers flatten the tissue, thereby reducing the diffusion distance for heat; 2) the substance or process used to cool the hammer, typically liquid nitrogen, is not applied directly to the muscle, so there is no vaporization at the interface with the muscle; and 3) the hammers provide a large heat sink. On the other hand, the hammers are somewhat slower, requiring at least several hundred milliseconds to lower the muscle chamber and accelerate the hammers. In addition, they cannot be used safely with rigid connections between the muscle and delicate instruments, such as force transducers. Although the hammers flatten the tissue, the final thickness is limited by the attachments at the ends of the tissue. Using three layers of 25-µm-thick aluminum foil T-clips wrapped around the muscle suggests that the absolute minimum thickness of the flattened tissue would be >100 µm, not much less than the minimum thickness of our muscle preparation. This consideration suggests that the flattening provided by hammers does not confer a great benefit when thin tissue is studied. Other relevant comparisons relate to the coolant and the freezing rates.
Coolant.
The availability and ease of using liquid nitrogen and dry ice make
these the much preferred methods of cooling. To our knowledge, there
are no coolants with melting points below that of liquid nitrogen and
boiling points above 37°C, and there are only two readily available
substances that remain liquid at very cold temperatures and do not boil
at room temperature, isopentane and acetone. Isopentane freezes at a
substantially lower temperature than the sublimation point of the dry
ice used here (160°C vs. 78°C) but boils at a temperature
(28°C) just below normal body temperature. It could probably be used
effectively to freeze tissue beginning at 37°C because very little
gas would form before the tissue temperature fell to its boiling point.
The reason for using acetone in the present experiments is that taking
advantage of isopentane's lower melting point requires extra care to
keep it liquid. If dry ice were used to chill the coolant, no advantage
would be gained. If liquid nitrogen is used, a buffer must be provided
to prevent freezing of the coolant to be injected. A simple buffer
might be derived by freezing some of the coolant with liquid nitrogen and then using a slurry of liquid and frozen isopentane to chill the
tube containing solution to be injected. (Attempting to inject the
slurry directly would probably plug the tubes.) We did not use this
method, however, because the results presented above suggested that
adequate freezing rates could be obtained with this agent. In addition,
the greater volatility of isopentane caused greater air pollution in
the laboratory. Overall, these considerations suggested that the best
balance of simplicity and speed was achieved with acetone chilled by
dry ice.
Speed of cooling.
When freezing at 0°C is used to estimate the point at which reactions
of interest would be stopped, the calculations in Fig. 3 suggest that
the hammers cooled with liquid nitrogen would stop the reactions in
about the same time as isopentane at 160°C. The nearly identical
rates occur because the surface of the aluminum is warmed nearly
instantly to ~160°C by the muscle. Whether freezing rates with the
two methods would be identical depends on the extent to which the ideal
circumstances assumed in the calculations can be achieved.
Deviations from the ideal. The calculations in the APPENDIX assume that the cooling substance reaches the muscle as specified, that there is a perfectly stirred boundary at the muscle surface for liquid coolants, and that there is an ideal heat transfer rate from tissue to coolant. In practice, these ideals are not achieved. There must be some warming as the coolant approaches the muscle, and this is likely to be much greater with liquid coolants that are warmed as they pass through tubing and chamber. It is also unlikely that coolant flow is fast enough to provide a perfectly stirred boundary, and the imperfections in the liquid boundary are probably greater than for the interface between tissue and cold aluminum. All of this suggests that metal hammers are likely to provide more rapid cooling than organic solvents, despite calculations to the contrary.
On the other hand, it should be mentioned that the authors who developed the cold-hammer freezing technique measured the speed of freezing with a thermal junction placed between two frog sartorius muscles, instead of a single squashed muscle, as used in the present calculations. Their muscles began at 0°C and the signal from the thermal junction began to deviate suddenly from the baseline ~80 ms after the hammers touched the muscle. This is substantially slower than the 8 ms calculated to freeze a 200-µm-thick slab of muscle in Fig. 3. Because the time taken to freeze the tissue increases approximately with the square of the distance from the surface, a tripling of the thickness of the double muscle arrangement would be required to increase the time to the 80 ms calculated here. This estimate is a little larger than expected, but not completely unlikely, particularly when the thickness of the thermal junction and its coating are considered. In conclusion, we describe a method of using automated perfusion of coolant for freezing tissue rapidly in preparation for assay of reactants. Calculations suggest that, if a perfectly stirred boundary can be maintained, freezing rates will approach those achieved with cold metal hammers. Assessment of the lag between the first appearance of smooth muscle phosphorylation and the onset of force generation further suggest that the time taken to stop the biochemical reaction is <200 ms. Further comparison of the rapid rise of phosphorylation and the much slower rise of force indicates that physiological events are likely to intervene between phosphorylation and force generation and that these are the principal cause of the lag in appearance of the two physiological parameters.| |
APPENDIX |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Two complexities of the cooling process greatly complicate mathematical descriptions of the freezing process: 1) heat capacities and thermal conductivity coefficients vary substantially over the temperature ranges considered, and 2) a thermal buffer at 0°C in the slab of tissue at the boundary between frozen and unfrozen tissue impedes heat flow between the two tissue zones. This buffering occurs because there is no temperature gradient across the slabs of tissue at 0°C. To achieve numerical solutions, the experimental situations were modeled by dividing the tissue into thin slabs and calculating the heat transfer between adjacent slabs. For liquid coolant freezing, there was assumed to be a stirred boundary at the interface between tissue and coolant, such that the tissue surface was held at the temperature of the coolant. For freezing with aluminum hammers, the aluminum was also divided into slabs, and the flow of heat from the boundary into the hammer was computed.
The thermal conductivity coefficients and heat capacities of aluminum
and water at different temperatures plotted in Fig. 5 were used to calculate the heat
transfer rates between adjacent slabs of tissue and temperature changes
resulting from heat flow. The slabs of tissue and aluminum were assumed
to be very thin compared with the width of the tissue, so that edge
effects were ignored. The adequacy of the description was tested by
making slab thickness and time increments progressively smaller until there was no difference in the computed results (Euler's method).
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The temperature profiles at 1.5- or 0.6-ms time increments are plotted
in Fig. 6 for the three experimental
situations considered. In addition to providing the freezing rates
plotted in Fig. 3, these profiles show that the aluminum hammers warm
to approximately the melting point of isopentane, so that the
theoretical cooling and freezing rates achieved with isopentane would
be approximately the same as with aluminum hammers. As indicated in the
DISCUSSION above, however, deviations from the ideal are
likely to be greater with the present method, suggesting that, in
practice, cold hammers are likely to be faster than liquid
coolants.
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ACKNOWLEDGEMENTS |
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Present addresses: R. Maass-Moreno, Dept. of Nuclear Medicine, National Institutes of Health, Bldg. 10, Rm. IC401, Bethesda, MD 20892; T. Burdyga, Dept. of Physiology, Liverpool Univ., Liverpool L69 3BX, UK; C. Y. Seow, Dept. of Pathology and Laboratory Medicine, St. Paul's Hospital, Univ. of British Columbia, Vancouver, BC, Canada V6Z 1Y6.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. E. Ford, Krannert Institute of Cardiology and Dept. of Medicine, Indiana Univ. School of Medicine, Indianapolis, IN 46202 (E-mail: lieford{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 November 2000; accepted in final form 8 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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), with superimposed values of myosin
light chain phosphorylation. Force record is signal-averaged tetani
from 7 muscles. Each force point in each muscle was the average of 67 data points digitized at 1-ms intervals (4 60-Hz cycles) to reduce
high-frequency noise, and force points are plotted at the midpoint of
the 67-ms interval. As indicated, force did not deviate from the
baseline until the 430-ms point and rose much more slowly than
phosphorylation (
).
